Variation of mesophyll conductance mediated by nitrogen form is related to changes in cell wall property and chloroplast number

Abstract Plants primarily incorporate nitrate (NO3−) and ammonium (NH4+) as the primary source of inorganic nitrogen (N); the physiological mechanisms of photosynthesis (A) dropdown under NH4+ nutrition has been investigated in many studies. Leaf anatomy is a major determinant to mesophyll conductance (gm) and photosynthesis; however, it remains unclear whether the photosynthesis variations of plants exposed to different N forms is related to leaf anatomical variation. In this work, a common shrub, Lonicera japonica was hydroponically grown under NH4+, NO3− and 50% NH4+/NO3−. We found that leaf N significantly accumulated under NH4+, whereas the photosynthesis was significantly decreased, which was mainly caused by a reduced gm. The reduced gm under NH4+ was related to the decreased intercellular air space, the reduced chloroplast number and especially the thicker cell walls. Among the cell wall components, lignin and hemicellulose contents under NH4+ nutrition were significantly higher than those in the other two N forms and were scaled negatively correlated with gm; while pectin content was independent from N forms. Pathway analysis further revealed that the cell wall components might indirectly regulate gm by influencing the thickness of the cell wall. These results highlight the importance of leaf anatomical variation characterized by modifications of chloroplasts number and cell wall thickness and compositions, in the regulation of photosynthesis in response to varied N sources.


Introduction
Nitrogen (N) plays a crucial role in facilitating plant growth and development.In an agricultural ecosystem, the rational application of nitrogen fertilizer may be an efficient strategy to increase nitrogen use efficiency and decrease environmental pollution [1].Higher plants utilize N from the soil mainly in the forms of ammonium (NH 4 + ) and nitrate (NO 3 − ) [2].However, plant N form preference and uptake rates of different N forms are speciesspecific.Some plants, such as rice and tea plants showed strong preference to NH 4 + ; whereas other species, such as Arabidopsis thaliana prefer NO 3 − [3].Even for NH 4 + -preferred plants, a supplement of NO 3 − to NH 4 + nutrition can lead to a better growth performance than plants supplied with NH 4 + nutrition alone [4].
When NH 4 + is supplied as the dominant or the only N source, plant growth is often inhibited, manifested by a reduced leaf area, relative growth rate and photosynthesis [2].Over the past years, numerous studies have observed the inf luence of N form on leaf photosynthesis [5][6][7], but the mechanisms have not been fully understood.Mesophyll conductance (g m ), stomatal conductance (g s ), and biochemical capacity have been demonstrated to be three major limitations to leaf photosynthesis [8], and CO 2 diffusion capacity contributes 35%-70% of total limitation to photosynthesis although the contribution can vary significantly at different environments [9].However, it is not known which limitation is the major determinant to the response of photosynthesis to N form.
g m represents the diffusion conductance of CO 2 from substomatal cavities to carboxylation sites inside chloroplasts.g m is a trait with high plasticity, and environmental stimuli including light intensity, water availability, salinity, and temperature can significantly affect g m [10][11][12][13].There is much evidence showing that g m can vary substantially among different nutrient supplements [14][15][16][17].For example, potassium deficiency can lead to more than 50% decline of g m in Brassica napus leaves when comparing with normal potassium supplement [14].Xiong et al. [17] indicated that g m is sensitive to leaf N status, and the value of g m increased with leaf N content.The importance of N source on g m has also been intensively studied.Generally, leaf N content was positively correlated with net photosynthetic rate (A) and g m [18].Nevertheless, the response of g m to different N forms exhibited variability in previous studies.Gao et al. [19] and Li et al. [20] showed a higher g m under NH 4 + conditions than that under NO 3 − conditions in rice when subjected to drought stress.In contrast, Liu et al. [16] observed a lower g m in female Populus cathayana when utilizing Table 1.Effects of different N forms on leaf photosynthetic characteristics in Lonicera japonica Treatment A (μmol m −2 s −1 ) g s (mol m −2 s −1 ) g m (mol m −2 s −1 ) V cmax (μmol m −2 s −1 ) J max (μmol m −2 s −1 ) C i (μmol mol −1 ) C c (μmol mol − + alone, a mixed N supply, and NO 3 − alone, respectively.Data are means ± SE (n = 5).The data followed by different letters are significant at P < 0.05 level.

NH 4
+ as the sole N source.The regulatory mechanism underlying the relationship between g m and N form remains unknown.Leaf anatomical traits, including chloroplast surface area exposing to the intercellular air spaces (S c /S) and the thickness of cell wall (T cw ), are major determinants to g m [21].g m is positively correlated with S c , while g m decreases substantially as T cw increases [22,23].A meta-analysis by Huang et al. [24] suggested that T cw shows great potential for enhancing leaf photosynthesis because of the large impact of T cw on g m and photosynthesis.The primary components of plant cell wall consist of cellulose microfibrils, along with a matrix of hemicelluloses, pectin, lignin, and structural proteins [25].There are many studies reporting the large responses of cell wall characteristics to environmental stimuli, including drought, salinity, and temperature [26][27][28].Transcriptomic analysis has highlighted a pivotal inf luence of N form on cell wall synthesis [29], which has been concluded from the co-expression of nitrate transporters and enzymes relating to cell wall synthesis.Unfortunately, the effects of inorganic N form on cell wall thickness and compositions are not fully understood.
To the best of our knowledge, there are merely two research studies indicating the potential impact of N form on cell wall properties.Podgórska et al. [30] found that, in comparison with NO 3 − fed Arabidopsis, NH 4 + fed plants possessed more densely assembled cellulose microfibrils and thicker cell walls, which in turn increased cell wall stiffness and inhibited plant growth.Głazowska et al. [31] proposed that NO 3 − can stimulate cellulose while impeding lignin accumulation in Brachypodium distachyon; furthermore, structures of hemicellulose and pectins had been found to be strongly inf luenced by N form, and NO 3 − can induce changes in the substitution pattern of xylan and result in reduced esterification level of homogalacturonan.However, the direct relationship between cell wall compositions and g m were not addressed here.The inf luence of N form on the structure of cell wall and compositions can potentially affect photosynthesis via g m .
Lonicera japonica, commonly known as Honeysuckle, is a common shrub belonging to the Caprifoliaceae family [32], and it is an important Chinese medicinal material for its anti-inf lammatory effect on viral infections [32].In our previous study, L. japonica showed a preference to NO 3 − in comparison with NH 4 + , which can be revealed from the rapid growth rate under NO 3 − nutrition [33].
However, it is not known whether N form can substantially affect leaf photosynthesis in L. japonica.In this study, L. japonica was hydroponically grown with three N forms (NH    treatment than that in the other two treatments (Table 1).In accordance with the lower C c under NH 4 + nutrition, the greater depressions observed in g s and g m , compared to V cmax and J max suggested that the decline of CO 2 diffusion conductance, especially of g m , was the major reason for the lowered photosynthesis in NH 4 + nutrition than the other two N forms.This conclusion can also be drawn from the relative limitation analyses (Fig. 1).In general, the relative limitation of mesophyll diffusion on photosynthesis (l m ) was larger in NH D chl-chl , the distance between adjacent chloroplasts; f ias , the volume fraction of intercellular air space; L chl , the distance between chloroplasts and cell walls; N chl1 , chloroplast numbers in spongy parenchyma per mesophyll cell profile; N chl2 , chloroplast numbers in palisade cell per mesophyll cell profile; Sc/S, chloroplast surface area exposed to intercellular air space per leaf area; Sc/Smes, chloroplast surface exposed to mesophyll cell surface area per leaf area; Smes/S, mesophyll cell surface area exposed to intercellular airspace per unit leaf area; Tcw, cell wall thickness.A, AN, and N represent N forms of NH 4 + alone, a mixed N supply, and NO 3 − alone, respectively.A, AN, and N represent N forms of NH 4 + alone, a mixed N supply, and NO 3 − alone, respectively.Data are means ± SE (n = 4).The data followed by different letters are significant at P < 0.05 level.

Effect of different N forms on leaf morphological and anatomical traits
Compared to NO 3 − and the mixed N supply, NH 4 + nutrition significantly decreased L A of the newly expanded leaves (Table S1, see online supplementary material), and N form had no substantial impact on leaf mass per area (M A ), thickness (T L ), or mesophyll thickness (T mes ).In contrast, leaf ultrastructural characteristics were significantly affected by N form (Table 2 and Fig. 2).Obviously, mesophyll cells tended to be stacked together in plants fed with NH 4 + nutrition and the starch granules were significantly accumulated in the chloroplasts (Fig. 2).Mesophyll cell surface area exposed to intercellular airspace (S mes /S), S c /S, chloroplast surface exposed to mesophyll cell surface area (S c /S mes ), as well as the volume fraction of intercellular air space (f ias ) were notably reduced in NH 4 + treatment than those in the other two N forms; while T cw and distance between adjacent chloroplasts (D chl-chl ) were significantly higher under NH 4 + treatment.N form had no significant effect on chloroplast length (L chl ), chloroplast thickness (T chl ), or distance between chloroplasts and cell walls (L cyt ), which suggested that chloroplast size was independent on N form and that the variations of S c /S and S c /S mes may relate to a different chloroplast number (Table 2; Table S2, see online supplementary material).
Indeed, the chloroplasts number in each cross section of spongy parenchyma and palisade cell was largely more decreased in NH 4 + treatment than that in the other two N forms (Table 2), and chloroplast number was positively correlated with S c /S and S c /S mes (Fig. S2, see online supplementary material).Moreover, g m exhibited a positive correlation with S c /S, S c /S mes and f ias , and a negative correlation with T cw and D chl-chl (Fig. 3).However, g m did not show any significant correlation with S mes /S.These results suggested that the variation of g m among the three N forms was related to the changes of leaf anatomical characteristics.This conclusion was also supported by the positive correlation between g m -Harley and g m -anatomy, which was calculated with leaf anatomical characteristics using gas diffusion model proposed by Tomás et al. [34] (Fig. S3, see online supplementary material).

Key structural factors regulating a and g m
Considering the different components along mesophyll diffusion pathway, the percentage limitations of g m were estimated.Liquidphase CO 2 resistance was the predominant factor determining g m (more than 90%) under different treatments (Fig. 4a), as CO 2 diffusion rate in water was much slower than that in air.Among the components that were responsible for liquid-phase limitations, the stroma had the largest impact (55%-62%) on g m , but it was not statistically different among the treatments.In contrast, it seems that cell wall was the pivotal factor that differentiates the treatments, the cell wall resistance in NH 4 + treatment was 56.3% larger compared to NO 3 − treatment (Fig. 4b).Taken together, the impact of different forms of N on leaf morphological and anatomical structural factors were summarized in a schematic diagram (Fig. 5).

N form had significant effects on cell wall compositions
In the present study, dynamic sampling of leaves fed with different N forms allowed us to investigate the detailed alterations in cell wall composition.Leaf photosynthesis and the cell wall components were continuously monitored since the start of N form treatment at an interval of 7 days (Fig. 6).Significant changes in absolute concentrations of cell wall compositions were detected since day 7.In general, NH 4 + reduced cellulose content but increased hemicellulose level; nonetheless, N form supply did not have any significant effect on pectin abundance.Lignin content was significantly increased in NH 4 + nutrition at 28 days after treatments, compared with NO 3 − and mixed N treatments.Concerning the temporal variation in cell wall compositions among treatments, the relative abundance of each component was calculated based on day 0 (100%) (Fig. 6).The application of NO 3 − and mixed N treatments resulted in a gradual increase of the relative abundance of all cell wall compositions since 7 days after the start of N form treatment.Whereas NH 4 + supplement led to large f luctuations in the levels of cellulose and hemicellulose on day 7, where its relative abundance was statistically different from treatments of N and AN.
The relative abundance of lignin was notably higher compared to the N and AN treatments since day 14.g m was positively correlated with cellulose concentration (Fig. S4, see online supplementary material, R 2 = 0.46, P < 0.01), whereas it exhibited a negative correlation with both hemicellulose (Fig. S4, see online supplementary material, R 2 = 0.49, P < 0.01) and lignin (Fig. S4, see online supplementary material, R 2 = 0.57, P < 0.001).However, g m was not correlated with pectin.

Path analysis of the correlations between leaf anatomical characteristics and leaf photosynthesis
N form had a significant effect on leaf cell wall compositions, T cw , f ias , and chloroplast numbers.Path analysis indicated that S c /S (R 2 = 0.91, P < 0.05) and T cw (R 2 = 0.92, P < 0.01) had direct effects on g m (Fig. 7).Variation of S c /S was mainly related to chloroplast number in mesophyll cells, although the variation of S mes /S may affect S c /S. Consistently, N form had a larger effect on S c /S mes than S mes /S (Table 2).T cw was related to cell wall compositions, and the relationship was mainly driven by the variations of lignin and hemicellulose.Notably, the path analysis did not show a direct inf luence of cell wall compositions on g m (Fig. 7).

Discussion
The decline of leaf photosynthesis under NH 4

supplement is mainly related to decreased CO 2 diffusion
In the present study, we found that NH 4 + treatment had led to a substantial reduction in leaf A of L. japonica (Table 1).The declines of g s and, in particularly, g m are the major causes for the depressed  leaf A, although V cmax and J max are also significantly reduced in NH 4 + treatment (Table 1).The dominating effect of CO 2 diffusion capacity in responses of photosynthesis to N form has also been found in previous studies [16,19,20].The mechanisms relating to the inf luence of N form on g m will be discussed later.
Here, a lower g s in NH 4 + supply was observed when comparing with the other two N forms (Table 1).The inhibition of g s by NH 4

+
nutrition has been commonly reported in previous studies [35,36], the lowered g s under NH 4 + supply is suggested to be a strategy to reduce NH 4 + transportation into leaves [35].Generally, g s is regulated by stomatal anatomical features, and the reduction of stomatal aperture and stomatal number per unit leaf area would negatively affect g s [37].The impression of leaf indicates that L. japonica absorbs water and CO 2 across hypostomatous leaf (Fig. S5, see online supplementary material).When the three treatments were compared, NO 3 − supplement had a comparable stomatal density with NH 4 + , while the stomatal length and width were significantly higher than that of NH 4 + supplement (Table S3, see online supplementary material).Guo et al. [38] reported that the Arabidopsis mutant with a low-level expression of nitrate transporter gene AtNRT1.1 possessed a lower g s , indicating that nitrate may play a role in stomatal opening.However, further research is necessary to explore the mechanisms underlying the regulation of N form on stomatal aperture.Leaf biochemical functions, namely V cmax and J max , are determined by Rubisco content and activity and are associated with both the N content and N allocation of leaves [24].In the present study, the decreased biochemical functions in NH 4 + supply were accompanied by a higher leaf N concentration per leaf area (N a ) as well as the Rubisco content (Table S4, see online supplementary material); similar results were observed in Cao et al. [39].Compared with NH 4 + treatment, the NO 3 − and mixed N treatment had higher Rubisco activity, which might account for the increased V cmax and J max (Table 1; Table S4, see online supplementary material).Why NO 3 − supplement would increase Rubisco activity of L. japonica remains unclear, however.One possible explanation may be the chloroplast C c accumulation in a condition of NO 3 − promoted the activation of Rubisco [40] (Table 1).Moreover, the distribution of leaf N is crucial in determining A, by affecting N investment on photosynthetic apparatus (e.g., Rubisco enzyme) or non-photosynthetic apparatus (e.g., cell wall) [41,42].In comparison with NO 3 − and mixed N treatments, NH 4 + supplied plants possessed a lower photosynthetic N use efficiency (PNUE) (Table S4, see online supplementary material).According to Xue et al. [43], most of the PNUE variation could be explained by Rubisco N allocation fraction and g m .The inf luence of N form on N allocation has been reported in Leymus chinensis [44], and this differential leaf N allocation between N form may serve as a plant strategy to acclimate NH 4 + stimuli.
g m reduction under NH 4 + supplement is mainly related to declined S c /S and thicker cell walls It has been frequently found that both S c /S and cell wall thickness serve as the main leaf anatomical factors that determine the largest g m [10][11][12].The results obtained from path analysis also suggested that the lower g m in NH 4 + treatment was mainly caused by the declined S c /S and the thicker cell walls (Fig. 7), although the lower f ias in NH 4 + treatment may restrain gas-phase CO 2 diffusion (Table 2, Figs 4 and 5).
The value of S c /S is related to the arrangements of both mesophyll cells and chloroplasts (Fig. 5).Considering the little difference of S mes /S among three N forms, the decreased S c /S in NH 4 + treatment was mainly related to its lower S c /S mes , which was in turn related to less chloroplasts in mesophyll cells in NH 4 + treatment (Table 2 and Fig. 5; Fig. S2, see online supplementary material).Indeed, chloroplast development can be significantly affected by N form.Findings by Ariga et al. [45] revealed that Arabidopsis mutants having the lower expression of HOMEOBOX PROTEIN (a transcription factor depending on nitrate supply) possessed fewer chloroplasts in mesophyll cells.Similarly, An et al. [46] showed that overexpression of PdGNC, which is a nitratedependent GATA transcription factor, can lead to a significantly increased chloroplast number and photosynthesis in Arabidopsis.Therefore, N form can affect g m and leaf photosynthesis by regulating chloroplast development (Fig. 7; Fig. S2, see online supplementary material).It has been suggested that a larger photorespiration rate can lead to a lower apparent g m because of the photorespiratory CO 2 release and refixation [47].In the present study, the variations in photorespiration f lux partially represented by Γ * may inf luence the observed g m (Table S5, see online supplementary The magnitude of this effect depended largely on the effective location of CO 2 release from (photo)respiratory in some cases [48]; for instance, the smaller D chl in NO 3 − would force the (photo)respired CO 2 to release through chloroplasts rather than mixed with CO 2 coming from the intercellular space, and the later would enlarge the CO 2 diffusion resistance from cytosol (r cyt ) and chloroplast envelope (r env ) (Table 2 and Fig. 5).Despite the possibility for a large impact of photorespiration on g m being low, because we have observed a correlation between g m -anatomy and g m -Harley (Fig. S3, see online supplementary material), we cannot exclude this possibility.Cell walls are major restraints for CO 2 diffusion inside leaves (Figs 4 and 5).It is suggested that cell wall resistance contributes 25%-50% of the overall mesophyll resistance [12].The variation of the contribution is related to the effective porosity of cell walls [22], which cannot be directly measured due to methodological limitations.In the present study, T cw was significantly higher in NH 4 + nutrition compared to NO 3 − nutrition (Table 2), which is one of the reasons for the declined g m and A in NH 4 + nutrition (Figs 3 and 7).However, the inf luence of N form on T cw was species dependent.Gao et al. [19]

The influence of N form on cell wall compositions
Plant cell walls are reported to be susceptible to environmental perturbations and can respond very quickly to better cope with these changes [27,49].Similarly, we found that N form can significantly affect cell wall compositions in the present study.Generally, cellulose content was increased while hemicellulose and lignin were reduced in NO 3 − nutrition (Fig. 6).The inf luence of N form on cell wall compositions may relate to the changing activity of critical enzymes.Landi and Esposito [29] found the co-expression of NO 3 − transport and cellulose synthesis genes in Arabidopsis, which suggested a connection between nitrate assimilation and cellulose synthesis.In contrast, it has been found that nitrate leads to the down-regulation of a majority of genes involved in phenylpropanoid pathways, which will in turn result in the decrease of lignin content [30,31].Consistent findings reported by Głazowska et al. [31] indicated that the absence of a difference in lignin content among different N forms on early stage could be related to less tissue NO 3 − accumulation in the mixed N and NO 3 − treatments (Fig. 6).Furthermore, the pectin content was independent from N forms; the different responses patterns of pectin are assumed to be species dependent (e.g., succulent plants, [27]) or environment dependent [26].
The studies relating to the correlation between cell wall compositions and g m are amounting, but the results are inconsistent.For instance, the multi-species analysis showed the variations in hemicelluloses and cellulose were negatively related with g m [49]; other studies demonstrated a key role for pectin in determining g m [21,28].The direct inf luence of cell wall compositions on g m needs to be further studied.Most importantly, results of the recent studies may indicate the coordinated modifications in the composition and the thickness of cell wall in setting g m [28,29].Similarly, the path analysis did support the inf luence of cell wall compositions on g m by changing T cw (Fig. 7).According to Carriquí et al. [49], the opposite relationship involving different phytogroups between T cw and the absolute abundance of hemicellulose was identified.Hemicellulose is a heterogeneous polysaccharide, which plays a crucial role in strengthening cell walls by connecting cellulose microfibrils and attaches them to lignin [50].An in situ study of wood structure revealed a positive relationship between the dynamic dissolution of lignin and hemicellulose and the T cw reduction rate, which would facilitate better transportation of active compounds deep into the cell wall [51].The decrease in the lignin and hemicellulose contents of the cell wall may thus be a key aspect involved in promoting CO 2 diffusion in NO 3 − supplement, by reducing T cw (Fig. 6 and 7; Fig. S4, see online supplementary material).

Conclusion
In summary, leaf photosynthesis of L. japonica was decreased in NH 4 + -fed leaves more than that in NO 3 − nutrition.Limitation analysis revealed that the variation of g m explained most of the reduction of photosynthesis under NH 4 + treatment, followed by g s and biochemical capacity.Variations of g m under different N forms primarily correlated with changes in leaf anatomical traits of T cw and chloroplast numbers, and cell wall was thicker and chloroplast number was lower in NH 4 + -fed leaves compared to NO 3 − nutrition.Furthermore, cell wall compositions were substantially affected by N form, and lignin and hemicellulose were the pivotal cell wall components in regulating g m among N form.Compared to NO 3 − , the decreased g s in NH 4 + condition was mainly mediated by the decrement of stomatal aperture, while the reduction of biochemical capacity in NH 4 + is primarily associated with the differences in nitrogen allocation and the decrease of Rubisco activity.This research emphasized the significance of leaf anatomical changes in regulating g m and leaf photosynthesis among different N forms, and further research is required to explore the mechanisms underlying the association between N assimilation and the synthesis of cell walls.

Plant material and treatment
The experiments were carried out in the intelligent greenhouse of Nanjing Agricultural University, China.Seedlings of L. japonica were maintained in the greenhouse with a day/night temperature of 28 • C/18 • C. Light intensity was maintained at 1000 μmol (photons) m −2 s −1 at the leaf level with SON-T AGRO 400 W bulbs, and the photoperiod was set to 14 h light/10 h dark.Before treatment, seedlings were supplied with a half-strength nutrient solution for 2 weeks and full-strength nutrient solution for another 2 weeks for adaption (for nutrient solution composition, see Table S6, see online supplementary material).Homogeneous seedlings were then selected for the N form treatment.The provided N concentration was 2.8 mM, and it was supplied in three distinct N forms: (NH 4 ) 2 SO 4 alone, Ca(NO 3 ) 2 alone, and a combination of 50% (NH 4 ) 2 SO 4 and 50% Ca(NO 3 ) 2 .CaCl 2 was added to the NH 4 + and mixed treatments to adjust Ca concentration to the level of the NO 3 − treatment (2.8 mM).

Gas exchange and chlorophyll fluorescence measurement
Four weeks after treatments started, leaf gas exchange and chlorophyll f luorescence of new fully-expanded leaves were simultaneously measured with a LI-6800 portable photosynthetic analyser equipped with a 2 cm 2 f luorometer chamber.The leaf temperature with the chamber was 25 ± 0.5 • C, the relative humidity was maintained at 40-60%, air f low rate was 500 μmol s −1 , CO 2 concentration (C a ) was 400 ± 6 μmol mol −1 , and the photosynthetic photon f lux density (PPFD) was set as 1000 μmol photons m −2 s −1 , and the light quality was set to10/90 of blue/red light.When gas exchange had stabilized for at least 20 min, A, g s , C i , steady-state f luorescence yield (F s ), and the maximum f luorescence (F m ) were measured simultaneously with a multiphase f lash.The effective quantum efficiency of photosystem II (Φ PSII ) was quantified as Φ PSII = (F m -F s )/F m .Five individual replicates were conducted for each treatment.For photosynthetic CO 2 responses (A/C i ) measurement, the same environmental conditions within the leaf chamber were controlled.After attaching the leaves to the leaf chamber for 20 min, C a was set in a descending series of 400, 350, 300, 250, 200, 150, 100, 400, 450, 500, 550, 650, 800, and 1000 μmol CO 2 mol −1 .Each CO 2 concentration was allowed to equilibrate for approximately 2-3 min.The V cmax and J max were calculated using Sharkey et al.'s [52] model.CO 2 leakage through the gaskets was inevitable in gas exchange measurement [53].To reduce the effect of leakage, leaks were checked when the CO 2 concentration within the leaf chamber was 800 and 1000 μmol CO 2 mol −1 , to ensure no more than 0.2 μmol CO 2 mol −1 leaked.When stable photosynthetic conditions were reached, the data were recorded after CO 2 concentration and water vapour between the leaf and the reference chamber were automatically matched [54].

Estimation of g m using the variable J method
Both g m and C c were estimated according to Harley et al. [55]: where the photosynthetic electron transport rate (J) was calculated as J = Φ PSII × PPFD × α × β.In this context, α represents the absorption of light by leaf, while β denotes the fraction of quanta absorbed by PSII.The product of α × β was estimated according to Valentini et al. [56], from the relationship between Φ PSII and Φ CO2 (quantum efficiency of CO 2 fixation).Because no substantial variation in α × β was observed across the three treatments (Table S5, see online supplementary material), the average α × β value of 0.42 was used to calculate g m in all three treatments.The apparent intercellular CO 2 compensation point of net CO 2 assimilation rate in the absence of respiration (C i * ) and day respiration rate (R d ) were measured according to the method proposed by Brooks and Farquhar [57].The methods relied on measuring the A/C i relationship at three different PPFDs (150, 300, and 600 μmol photons m −2 s −1 ) with each having four ambient CO 2 concentration of 25, 50, 75, and 100 μmol CO 2 mol −1 .Prior to initiating measurements, leaves were placed in a condition at a PPFD of 600 μmol photons m −2 s −1 and a CO 2 concentration of 100 μmol CO 2 mol −1 for 30 min for adaption.Their linear regressions were then predicted to converge at one point where the x-axis and y-axis of the point were defined as C i * and R d .
The CO 2 compensation point in the absence of mitochondrial respiration (Γ * ) was then calculated as follows: Values of Γ * , C i * , and R d in three treatments could be found in Table S5 (see online supplementary material).

Leaf area, leaf N content, and Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) content and activity
New fully-expanded leaf was randomly selected and photographed.The L A obtained from each image was calculated using Image-Pro Plus.Subsequently, leaves were dried at 105 • C for 15 min, and at 65 • C to reach a stable weight and samples were weighed.Leaf mass area (M A ) was calculated as: M A = leaf dry weight/L A .To determine per leaf area concentration of N (N a ), dried leaf samples were digested with H 2 SO 4 -H 2 O 2 at 280 • C, and an Auto-analyzer 3 digital colorimeter (AA3, Bran+Luebbe Inc., Norderstedt, Germany) was used to determine the N concentration according to the methods of Guo et al. [58].Content and activity of Rubisco were determined by Plant Rubisco Elisa Kit (Jiangsu Enzyme Free Biotechnology Co., Ltd, Nanjing, China).For each replication, 0.10 g leaf sample was grinded to homogenate in a pre-cooled mortar, with 1.0 mL chilled PBS solution (pH 7.2-7.4).The mixture was centrifuged at 8000 × g for 20 min and the supernatant was used for the determination following the manufacturer's protocol.

Quantitative limitation analysis of a
The relative limitations on leaf photosynthesis resulting from g s (l s ), g m (l m ), and biochemical capacity (l b ), where the values of l s , l m , and l b added up to 100%.According to Grass and Magnani [59]: where g tot represents the CO 2 conductance from the leaf surface to carboxylation sites inside the chloroplast, which can be determined through g sc and g m .∂A/ ∂C c was estimated according to the slope of the A/C c curves over a C c range of 50-100 μmol mol −1 .

Anatomical analysis
After the gas exchange measurements, 5 × 5 mm leaf sections were collected between the main veins from the same leaves for anatomical measurements.The preparation of paraffin and resin sections followed the method of Lu et al. [14] and Gao et al. [19].The safranin-fast green staining was applied to the paraffin sections, which were then observed using a Nikon Eclipse ci microscope equipped with a Nikon digital microscope camera.The images were captured at a 400× magnification to measure the T L , T mes , with ≥20 fields of view per treatment.Ultrathin resin sections were contrasted with 2% uranyl acetate and lead citrate, then viewed with transmission electron microscopy (TEM).Graphs were acquired at a direct magnification of 2000-8000× to measure T cw , L chl and T chl , D chl-chl , L cyt , and chloroplast numbers per mesophyll cell, with ≥30 fields micro-pictures for each treatment.All the images were analysed using Image-Pro Plus software.The leaf density (D L ) and the f ias was calculated using measurements from light-microscope graphs.D L and f ias were determined as follows: where M A represents the weight of specific leaf (mg cm −2 ), ΣS s denotes the combined cross-section area of mesophyll cells, and W corresponds to the width of the measured framed range.S c /S and S mes /S were calculated according to the method of Evans et al. [60] and Syvertsen et al. [61].
where the L mes is the total mesophyll surface length exposed to the intercellular air space; L c represents the chloroplast surface length facing the intercellular air space.The curvature correction factor F was calculated using the method of Thain [62], depending on the shape of the mesophyll cell.Brief ly, based on differences between palisade and spongy cell, we derived F as a weighted average from both spongy (F 1 ) and palisade (F 2 ) mesophyll distributions: where a and b represent the mean mesophyll length and mean mesophyll thickness.The eccentricity e and elliptical integral E were calculated as proposed by Weast [63]: For stomatal features measurement, the impression protocol was conducted for the assessment of leaf stomatal density and aperture (length and width) [64].Images were captured with an optical microscope system (Olympus IX71, Olympus Optical, Japan) at a magnification of 200× and 400× to measure the leaf stomatal density and at a magnification of 1000× for the measurement of the length and width of stomatal aperture, with ≥20 fields of view per treatment.

g m modelled from anatomical characteristics
The one-dimensional gas diffusion model modified by Tomás et al. [34] was used to determine g m , which is divided into leaf gas and liquid components: where g ias represents the gas-phase conductance from substomatal to the cell wall's outer surface; g liq denotes the liquid conductance from the cell wall's outer surface to chloroplasts.R is the gas constant, T k is the absolute temperature (K), and H represents Henry's law constant (2943.3Pa m 3 K −1 mol −1 for CO 2 ) [19].In this model, the gas-phase conductance (g ias ) depends on f ias and effective diffusion path in the gas phase (ΔL ias ).g ias was calculated as follows: where σ is the diffusion path tortuosity [19,34] and D a (m 2 s −1 ) is the diffusion coefficient for CO 2 in the gas-phase [34].ΔL ias is defined as half of T mes .
The g liq was determined by various mesophyll cell properties, including conductance in cell walls (g cw ), plasmalemma (g pl ), cytosol (g cyt ), stroma (g st ), and chloroplast envelope (g env ).Thus, g liq was defined as: g liq = S c r cw + r pl + r cyt + r en + r st S (17) where r cw , r pl , r cyt , r en , and r st are the reciprocal terms of g cw , g pl , g cyt , g env , and g st , respectively.Alternatively, according to Tholen et al. [65], CO 2 diffusion inside the cell differs, such that one route involves cell wall parts and chloroplasts (g cel,1 ), whereas the other route involves inter-chloroplast areas (g cel,2 ).The corresponding resistance was expressed as r cel,1 and r cel,2 .As a result, the equation for g liq was transformed into: g liq = S mes r cw + r pl + r cel,1 + r cel,2 S (18) The liquid-phase CO 2 diffusion pathway, whether for g cw , g cyt , or g st , is expressed as follows: where r f,i is a factor that accounts for the decreased CO 2 diffusion in the aqueous phase vs. free diffusion in water; this factor was set to 1.0 for the cell wall and 0.3 for the cytosol and stroma.ΔL i (m) represents the distance that CO 2 diffuse in the corresponding component, while p i (m 3 m −3 ) ref lects the effective porosity.
Evidence showed that the effective porosity varied with cell wall thickness [53].Therefore, a least squared iterative analysis was used by varying the p i of the cell wall to obtain the best fit value (highest R 2 ) between the measured and modelled g m value [34].As a result, p i was taken as 0.30.D w denotes the CO 2 diffusion coefficient in the aqueous phase.Both g pl and g env were set as 0.0035 m s −1 [60].

Quantitation of the anatomical limitation of g m
The factors inf luencing g m consist of both the conductance in gasphase (l ias ) and in liquid-phase (l i ).The l ias can be calculated as: The share of g m contributed by the cellular-phase conductance (l i ), an indicator of the limitation of the cell wall, cellular contents, as well as the plasmalemma, was determined as: where g i represents the conductance for diffusion in each corresponding pathway.

Cell wall compositions determination
Dynamic analyses of cell wall compositions were conducted using plant samples collected on four specific sampling days for each treatment: days 0 (before treatment), 7, 14, and 28.Leaves were collected at the early morning (05:30-06:00) to minimize leaf starch content and 1.0 g of each leaf sample was boiled in absolute ethanol until the tissue became bleached.Alcohol-soluble residues were eliminated with acetone, then the samples were air-dried and homogenized, which yielded the alcohol-insoluble residue (AIR).The AIR was identified as the crude cell wall material and was used for analyses of cellulose, hemicellulose, pectin, and lignin.
In the first step, 3 mg of each AIR were hydrolyzed using trif luoroacetic acid and subjected to 121 • C for one hour.Subsequently, the mixture was centrifuged at 13000 × g.The supernatant containing non-cellulosic cell wall components was maintained at 4 • C for the quantification of pectin and hemicelluloses; the pellet was used for the determination of cellulose quantification.The same protocol was used to calculate the cellulose and hemicellulose contents via the phenol sulfuric acid method [66].For pectin quantification, a galacturonic acid calibration curve was carried out in accordance with Blumenkrantz and Asboe-Hansen [67].Lignin was quantified using 20 mg of AIR sample, which was processed via the acetyl bromide method [68].The spectrophotometer used for measurements was HBS-1096A (Shanghai, China).

Statistical analysis
Data analyses were conducted using SPSS 25.0 to perform oneway ANOVA.To determine significant differences between treatments, the least significant difference (LSD) test was employed at a significance level of P < 0.05.Graphical depiction and regression analyses were carried out using Origin Pro 2021.PLS-PM analysis was performed with the R package 'plspm' using R statistical software (v.4.0.2).

Figure 1 .
Figure 1.Relative limitation analyses of stomatal conductance (l s ), mesophyll conductance (l m ), and biochemical capacity (l b ) on photosynthesis under different nitrogen(N) forms.A, AN, and N represent N forms of NH 4 + alone, a mixed N supply, and NO 3 − alone, respectively.The limitation of l s , l m , and l b together add up to 100% at each treatment.Data are mean ± SE (n = 5).

Figure 3 .
Figure 3. Correlations of g m and chloroplast surface area exposed to intercellular air space per leaf area, S c /S (a), chloroplast surface exposed to mesophyll cell surface area per leaf area, S c /S mes (b), mesophyll cell surface area exposed to intercellular airspace per unit leaf area, S mes /S (c), the volume fraction of intercellular air space, f ias (d), cell wall thickness, T cw (e), and the distance between adjacent chloroplasts, D chl-chl (f).A, AN, and N represent treatment with NH 4 + alone (open triangles), a mixed N supply (closed circles), and NO 3 − alone (open squares), respectively.Data are fitted by linear regression (n = 4).

Figure 4 .
Figure 4. Anatomical limitations of mesophyll conductance (g m ) (a) and the share of overall g m limitation (b) by cell wall (cw), plasmalemma (pl), chloroplast envelope (env), stroma (st), and cytoplasm (cyt) under different N forms.The inset showed the anatomical limitations of g m and the share of the overall g m limitation by gas phase and liquid phase.A, AN, and N represent N form of NH 4 + alone, a mixed N supply, and NO 3 − alone, respectively.Data are means ± SE (n = 4).Different letters indicated a significance at P < 0.05 level.

Figure 5 .
Figure 5. Schematic diagram of Lonicera japonica leaf anatomical traits (left side) and CO 2 diffusion pathway under NH 4 + alone supply (right side).In the left side, S c /S was outlined with red lines and S mes /S was marked with yellow lines.The accurate S c /S estimations overlapped S mes /S lines, whereas they are displayed independently here for clarity.Chloroplasts were highlighted with blue.Decreased chloroplast numbers resulted in the reductions in S c /S.The intercellular spaces were pointed out with pink discontinuous lines.The tight arrangement of mesophyll cells under sole NH 4 + suppliesled to increased intercellular spaces, which exacerbated the reductions in S c /S and S mes /S.In the right side, the black folded lines represent the strength of CO 2 diffusion resistance into the cell from cell wall (r cw ), plasma (r pl ), cytoplasm (r cyt ), envelope (r env ), and stroma (r st ), while the r cw and r cyt which are marked with red indicated the values of this component differed among the treatments (P < 0.05).Black discontinuous lines indicated the hypothesized pathways that could partially inf luence the CO 2 diffusion and thereby g m .

Figure 6 .Figure 7 .
Figure 6.The absolute and relative abundance of cellulose (a), hemicellulose (b), lignin (c), and pectin (d) on day 0, day 7, day 14, and day 28 under different N forms.The histogram plot indicates the absolute abundance of cell wall components and the insert line chart represents the relative abundance of cell wall components compared with day 0. The amounts of specific cell wall components were expressed based on the amount of alcohol-insoluble residue (AIR).A, AN, and N represent the N form of NH 4 + alone, a mixed N supply, and NO 3 − alone, respectively.Data are means ± SE (n = 5).Different letters indicate a significance at P < 0.05 level.
Cc, chloroplast CO 2 concentration; C i , intercellular CO 2 concentration; gm, mesophyll conductance; gs, stomatal conductance; Jmax, maximum electron transfer rate; Vcmax, maximum carboxylation rate.A, AN, and N represent the N form of NH 4 A, net photosynthetic rate;

Table 2 .
Leaf anatomical characteristics of Lonicera japonica under different N forms

Treatment S mes /S (m 2 m −2 ) S c /S (m 2 m −2 ) S c /S mes (m 2 m −2 ) f ias (%) T cw (μm) D chl-chl (μm) L cyt (μm) N chl1 (No.) N chl2 (No.)
± 2.3a 0.162 ± 0.002c 0.84 ± 0.03b 0.19 ± 0.02a 16.9 ± 0.1a 6.7 ± 0.1a reported that T cw was larger in NH 4 + nutrition than that in NO 3 − nutrition, which agrees with the results in the present study.Similarly, Liu et al. [16] found a larger T cw in NH 4 + nutrition in female poplar; in contrast, T cw was lower in NH 4 + nutrition in male poplar.The mechanisms relating to the inf luence of N form on T cw are not known, but the difference in cell wall compositions among N form treatments may account for the differential T cw .